Differentiation of live and dead cells is an important challenge in microbial diagnostics. In the case of pathogenic microorganisms, the potential health risks are limited to the live portion of a mixed microbial population. Four physiological states of microbes can be distinguished in flow cytometry using fluorescent stains: 1) reproductively viable; 2) metabolically active; 3) intact; and 4) permeabilized cells. Depending on the conditions, all stages except the permeabilized cells can have the potential of recovery upon resuscitation and thus have to be considered potentially live. DNA-based diagnostics tend to overestimate the number of live cells because they will also measure the DNA from dead cells, due in part to the relatively long persistence of DNA after cell death (e.g. up to 3 weeks). DNA extracted from a sample can originate from cells in any of the four above mentioned physiological states including the dead permeabilized cells. Thus most DNA-based diagnostics can not distinguish between live and dead bacteria.
The ability to quickly and accurately distinguish between live and dead bacteria is needed by today's challenges of “super-bug” antibiotic-resistance bacteria and terrorist threats of bio-weapons. The standard method in the past has been to take a swab sample of the potentially contaminated area and grow the collected bacteria on a media plate. The resulting colonies were then identified and counted to determine the level of contamination. Growing bacteria on media plates is slow and assumes that the media furnishes the entire necessary nutrient for growth. It was possible for live bacteria not to be detected if the media was not correct for the bacteria or the bacteria were difficult to grow on artificial media.
Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth medium and to proliferate in liquid nutrient broths. These traditional, culture-based tests are time-consuming and can work poorly with slow-growing or viable, but non-cultivable organisms. They do not provide real-time results or timely information that is needed in applications such as industrial manufacturing.
Polymerase chain reaction (“PCR”) has also been used as tool for the quick detection of bio-threat and foodborne pathogens. However, PCR itself does not discriminate from DNA coming from live pathogens (harmful) or dead pathogens (harmless). Accurate determination of live, damaged or injured, and dead bacteria is important in microbiology detection to avoid false alarm. Dead bacteria present after processes such as pasteurization or disinfections might present no hazard but still can be detected by PCR. Injured cells are virulent and may or may not be detected by standard procedures. PCR offers a more rapid and sensitive method than culture-based techniques, but the major limitation is the lack of differentiating the DNA from live or dead bacteria. In food matrices and the environment, DNA can be very stable and persist for extended periods of time, and therefore, it is desirable to have DNA-based assays that can identify only viable organisms.
In the past, PMA (propidium monoazide) or other DNA intercalating chemicals (such as EMA (ethidium monoazide bromide) or Phenanthridium derivatives have been used to detect only viable bacteria by qPCR. These compounds, though, are expensive, toxic and demand care in handling disposal.